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1Neuroscience Discovery, Eli Lilly & Company; and 2Indiana University, School of Medicine, Department of Anatomy and Cell Biology, Indianapolis, Indiana
Submitted 15 November 2005; accepted in final form 21 January 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Freund and Antal 1988; Manseau et al. 2005; Sotty et al. 2003; Woolf 1991). Some of the cholinergic neurons also ascend to innervate diffusely many parts of the neocortex to subserve cognitive processing (see review by (Everitt and Robbins 1997; Gaykema et al. 1990; Gritti et al. 1997; Sarter and Bruno 2000). Both cholinergic and GABAergic MS/vDB neurons are capable of firing spontaneously in unique rhythmic clusters of spike bursts that pace an oscillatory theta burst frequency of 3–12 Hz in freely moving animals and at a lower theta frequency range of 3–5 Hz in anesthetized animals in vivo (Apartis et al. 1998; Vinogradova et al. 1998) to generate hippocampal theta electroencephalographic (EEG) activity (Bassant et al. 1998; Bland 2004; Bland and Oddie 2001; Leung 1998). In addition, theta activity can also be induced locally within the hippocampal network after direct muscarinic receptor stimulations as shown in hippocampal slice recordings (Bland et al. 1988; Chapman and Lacaille 1999; Fellous and Sejnowski 2000; Golebiewski et al. 1992; Konopacki and Golebiewski 1993; Konopacki et al. 1987; MacVicar and Tse 1989; Manns et al. 2003; Stewart and Fox 1989c; Vertes et al. 2004; Vinogradova 2001; Vinogradova et al. 1998; Williams and Kauer 1997). Thus although HIPP theta is paced by MS/vDB, theta rhythm can be induced independently within the HIPP. Functionally, hippocampal theta EEG is tightly linked to a range of higher cognitive processes, (e.g., working memory and long- and short-term memory) as well as attention and REM sleep phases in animals and humans (Kahana et al. 2001; Kirk and Mackay 2003; Kleshchevnikov 1999; Klimesch 1999; Siapas et al. 2005; Tesche and Karhu 2000; Wiebe and Staubli 2001).
A multitude of neuromodulators released from diverse afferents of different brain regions are known to modulate theta burst firing activity of MS/vDB neurons (e.g., (Alreja 1996; Alreja and Liu 1996; also see reviews by Bland and Oddie 2001; Vertes et al. 2004). One important input that has not been explored extensively is the midbrain dopamine input. The septum receives midbrain dopamine innervation that originates from the ventral tegmental area, and both D1 and D2 class receptors are found in the septum (Berlanga et al. 2005; Chapman and Lacaille 1999; Fallon and Moore 1978; Gaspar et al. 1985; Gaykema and Zaborszky 1996, 1997; Kalivas et al. 1985; Lamsa et al. 2005; Lindvall and Stenevi 1978; Milner 1991; Milner and Prince 1998; Onteniente et al. 1984; Toth et al. 1997; Yoder and Pang 2005). Previous studies have shown that systemic or intra-MS/vDB, but not intra-lateral septal or accumbens, injection of dopamine increases theta EEG activity in the hippocampus (Collu et al. 1980; Marrosu et al. 1997; Miura et al. 1987; Yamamoto 1988), whereas transient inactivation of VTA suppresses HIPP theta EEG (Orzel-Gryglewska et al. 2006; Yoder and Pang 2005). Moreover, biochemical studies, including those that performed in DA receptor deficient transgenic mice, found that systemic or local HIPP administration of D1/5 agonists, via D5 receptor activation, increases acetylcholine (ACh) efflux, or turnover, in dorsal hippocampus and dentate/CA3 field (Acquas et al. 1994; Berlanga et al. 2005; Day and Fibiger 1994; Hersi et al. 1995, 2000; Imperato et al. 1993; Laplante et al. 2004; Robinson et al. 1979). This ACh, in turn, can activate muscarinic receptor-dependent hippocampal theta activity (Brazhnik et al. 1993; Chapman and Lacaille 1999; Fellous and Sejnowski 2000; Keita et al. 2000; Lukatch and MacIver 1997; Teitelbaum et al. 1975; Vinogradova et al. 1993) and theta-dependent long-term changes in synaptic plasticity (Huerta and Lisman 1995; Lisman and Otmakhova 2001.)
Given the importance of D1 class dopamine receptors in memory processing mediated in the prefrontal cortex (see Seamans and Yang 2004), we attempted to find out whether D1/5 receptor activation can also modulate theta pace-making MS/vDB neuronal theta burst firing, which may mediate D1-dependent cognitive processes via acetylcholine release in the hippocampus. In the present electrophysiological study, we have recorded extracellular single-unit MS/vDB neurons and determined their spontaneous theta burst firing responses to systemic injection of the D1/5 agonist dihydrexidine. We chose this route of administration to mimic the clinical route via which pharmaceutics are usually administered routinely. Preliminary findings have been reported as an abstract (Fitch et al. 2004).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Six rats (275–350 g) were killed with a ketamine/xylazine/acepromazine cocktail (40 mg/kg ketamine ip) and perfused intracardially with 0.9% NaCl followed by 4% paraformaldehyde and 0.4% picric acid in phosphate buffer. The brains were removed and postfixed overnight in the same fixative. Serial (30 µm) coronal sections were then cut with a vibrating microtome (Leica). Free-floating sections were incubated with 3% (vol/vol) H2O2 in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 10 min to quench endogenous peroxidase activity. Sections were then washed three times in PBS and preincubated overnight in PBS containing 0.3% Triton X-100 and 1.5% normal rabbit-serum to facilitate complete penetration of reagents through the tissue. On the next day, sections were incubated at room temperature for 24 h in the same solution containing an antibody against tyrosine hydroxylase (1:1000, Pel-Freez). Afterward, sections were washed three times in PBS and then incubated with a biotinylated secondary antibody (1:250, rabbit-anti-sheep IgG, Jackson ImmunoResearch Labs) for 1.5 h at room temperature. After that, sections were rinsed three times in PBS and then incubated in avidin-biotin peroxidase complex (ABC, Vector Labs) for 1 h, rinsed three times again in PBS and two times in 0.05 M Tris-buffered saline (TBS) before the addition of 0.05% 3'-3'-diaminobenzidine tetrahydrochloride and 0.003% H2O2 in fresh TBS to reveal the peroxidase activity. After mounting to slides, sections were Nissl-counterstained with 1% methyl green to reveal the overall cellular profile. Sections were imaged under a microscope (Leica) with darkfield illumination. Images were captured and digitized with a SPOT camera and software (Diagnostic Instruments).
Animals and surgical procedures for single-unit recordings
Male Sprague-Dawley rats (270–400 g) were anesthetized with urethan (1.5 g/kg ip, Sigma) and surgically implantated with a jugular vein catheter for intravenous drug administration. The animal was mounted on a stereotaxic frame (Stoelting) in a flat skull position. Core temperature was monitored by a rectal probe and maintained at 37°C by a heating pad (Frederick Haer). Borosilicate glass micropipette electrodes (1.5 mm OD, 1.17 mm ID, Sutter Instruments) were pulled by a programmable Flamming/Brown P-87 microelectrode puller (Sutter Instruments) and then filled with 0.5% sodium acetate in 2% Pontamine Skyblue (Gurr, BDH). To gain access to the MS/vDB region, the micopippette was advanced by a single-axis Narishige MHW-40 hydraulic micromanipulator mounted on a Kopf stereotaxic holder. A burr hole was drilled on the skull with stereotaxic coordinate of A-P 0.2–0.4 mm, M-L 1.2 mm, and the dura was carefully removed to expose the cortical surface. The recording pipette enter the brain through this hole and was advanced at a 10° angle from midline to reach the target site co-ordinates of MS/vDB with A-P 0.2–0.4 mm anterior to bregma, L-M 0.1–0.3 mm lateral from midline, and D-V 5.8–7.5 mm from the cortical surface (Paxinos and Watson 1997).
A concentric bipolar stimulation electrode (NE-100, Rhodes Medical Instruments) was positioned at either the fimbria/fornix (FF) or the VTA. The stereotaxic coordinates for stimulating electrode placement were: FF: A-P –1.6 to –1.8 mm posterior to bregma; A-P 0.2–0.4 mm anterior to bregma, L-M 1.3–1.5 mm lateral from midline; and D-V 4.0 –4.4 mm from the cortical surface; VTA: A-P –5.2 mm posterior to bregma, M-L 0.8 mm lateral from midline, and D-V 8 mm ventral to the cortical surface.
Electrophysiological recordings
Extracellular single-unit activity was amplified by a Xcell-3 Plus amplifier (Frederick Haer). Single-unit activity was isolated using a window discriminator (Model 74-60-3, Frederick Haer). Amplified signals were digitized and multiplexed by an A/D converter (1401 mini, Cambridge Electronics Design, Cambridge, UK), sampled at 10 kHz by a PC-based computer using Spike 2 software (Version 5, CED), and stored for off-line analysis. Programmed (Master-8, A.M.P.I.) monophasic square single-pulse stimulation currents (0.2-ms pulse-width, 0.5–1.2 mA) were delivered to the fimbria/fornix (0.2-ms pulse width, 1 Hz) to identify antidromic responses from MS/vDB neurons that project to HIPP (MS/vDB-HIPP) neurons or to activate VTA DA neurons (0.2-ms pulse-width, 20 Hz, 2 trains of 40 pulses each train, and with each train delivered at 0.25 Hz) via an optically isolated stimulation unit (Isoflex, A.M.P.I.).
MS/vDB neurons that project to the hippocampus (MS/vDB-HIPP) were identified by their antidromic response to FF stimulation (Apartis et al. 1998). They are characterized by a fixed response latency and collision of the antidromic spikes with orthodromic spike (see Fig. 2). In most instances, rhythmically bursting MS neurons could be identified by response to a 10-s tail pinch by a hemostat, and in some neurons 10-Hz train stimulation of the VTA can induce a brief period of theta burst activity.
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Dihydrexidine HCl (DHX) (Sigma), atropine sulfate (Sigma; both at 10 mg/kg iv) and SCH23390 (Sigma; 0.1 mg/kg iv) was dissolved in double-distilled water. They were prepared fresh daily just prior to injection. Drug solutions were administered intravenously at a rate of (0.1 ml/min) via surgically implanted jugular vein catheter using a syringe. Only one MS neuron per animal was tested. The baseline spontaneous firing activity of MS neurons were monitored for 20–40 min before any intravenous drug administration. Spontaneous activity was monitored for an additional 10–30 min beginning 5 min postinjection.
Data analysis
Spontaneous firing of single MS cells were analyzed with CED Spike-2 software routines providing measures of firing rate and burst firing parameters. In addition, autocorrelograms were computed. A spike burst was defined as a minimum of two action potentials with a maximum interspike interval of 30 ms. The minimum interburst interval was set at 60 ms with the minimum burst duration set to 10 ms. The burst parameters measured included the mean number of spikes per burst, mean number of bursts, mean interspike intervals.
If bursts of action potentials occur as rhythmically recurrent events, then event correlation in the autocorrelograms computed by the Spike-2 software (version 5.12, CED) will show periodic sinusoidal-like density peaks. The number of density peaks reflects the frequency burst firing. The higher the amplitude of the density peaks, the more regular the recurrence of rhythmic burst activity across the time period analyzed. Autocorrelograms showing three to six density peaks across a 1-s event correlation autocorrelation plot is characterized as in theta firing mode characteristic of rats under urethan anesthesia (Apartis et al. 1998).
The total duration of occurrence of rhythmic theta activity was also analyzed over the entire recording period. Autocorrelation analyses were made every 60 s using the number of density peaks measured to extract the theta burst frequency for each of these 60-s epoch. The percent of time that the MS/vDB neuron is spent in theta mode (called "theta dwell time") before and after drug treatment was then plotted over time.
For statistical treatment of the electrophysiological data, a log transformation of the data were used to reduce the skewness and to stabilize the variations. Spike activity was analyzed by two-way ANOVA using cell and treatment as the factors. Tukey's post hoc test was used to compare treatment groups (JMP Statistical Discovery Software, SAS Institute, Cary, NC). A P < 0.05 was deemed statistical significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Zaborszky and Cullinan 1996) have used double labeling of an anterograde tracer (injected into the VTA) combined with TH immunostaining to show that the fine TH-stained fibers with less varicosities and originating from the VTA are putative dopaminergic axonal fibers. In the present study, under light microscopy, we found extremely sparse TH-stained axonal innervation of the MS/vDB, in contrast with the dense TH innervation of the lateral septum (e.g., in the intermediate lateral septal nucleus). Only when we switched to dark-field microscopy that we were able to detect the very fine TH fibers that innervate throughout the MS/vDB region (Fig. 1B). At higher magnification (x20–40), many of these vertically hanging network TH-axonal fibers in the MS/vDB region are extremely fine in diameter, and a large proportion of them also contain small beaded varicosities (Fig. 1C).
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Fifteen of the 40 MS/vDB neurons (37.5%) responded to antidromic stimulation of the fimbria/fornix (Apartis et al. 1998). Antidromic responses are characterized by their invariable fast onset latencies (2–4 ms) at threshold current FF stimulation. Moreover, a spontaneous active spike collided with the antidromic spike when it occurred at the critical latency up to two times the onset latency of the antidromic spike. The antidromic responses of MS/vDB neurons that project via the fimbria/fornix (FF) to the hippocampus are likely to be MS/vDB-HIPP neurons (Dutar et al. 1985a,b; Stewart and Fox 1989b). Nevertheless, it is not known whether MS/vDB neurons that are not antidromically activated by FF stimulation may also be MS/vDB-HIPP projecting cells because the stimulating electrode placement in the FF may not be optimally positioned to activate all the axons of MS/vDB-HIPP neurons in some animals. Hence, we may be underestimating the total number MS/vDB-HIPP projecting neurons.
Electrophysiological properties of MS/vDB neurons—responses to tail pinch and VTA train stimulation
MS/vDB neurons have been shown to be sensitive to tail-pinch noxious stimulus (Lamour et al. 1984). We used this method as a way to confirm MS/vDB cell identity. A 10-s clamp of the tip of the tail by a hemostat elicited a transient increase in firing discharge and initiated or accentuated on-going rhythmic burst firing. The patterns of rhythmic burst firing corresponded to prominent theta burst firing as detected in the spike density peaks in the autocorrelogram (Fig. 2, C–E). Because the mesolimbic dopamine neurons of the VTA also project to the septum (Fallon and Moore 1978; Gaspar et al. 1985; Gaykema and Zaborszky 1996, 1997; Lindvall and Stenevi 1978; Milner 1991; Milner and Prince 1998; Onteniente et al. 1984), we tested in several MS/vDB neurons the impact of the dopamine-releasing 20-Hz train stimulations of VTA on MS/vDB cell firing. Brief stimulus trains delivered to the VTA (20 Hz, 2 trains of 40 pulses, 2-s train duration, delivered at 0.25 Hz) evoked transient rhythmic burst firing in several previously nonbursting MS cells during the tetani (n = 3 / 4 tested). Autocorrelograms obtained during, but not before, the VTA train stimulation show a typical theta mode bursting response with the number of spike density peaks indicating a burst frequency of 
5 Hz (see Fig. 2, F–H).
Endogenous dopamine tone regulates theta burst firing in MS/vDB neurons in vivo
Early studies have shown that direct injection of DA into MS/vDB in rats elicits theta EEG activity in hippocampus (Miura et al. 1987). Because many MS/vDB cells we have recorded already show spontaneous periodic theta burst firing activity in the baseline, we sought to determine whether endogenous DA "tone" may already be playing a role in driving tonically the spontaneous theta burst firing patterns of these MS/vDB neurons. Intravenous administration of a D1/5 antagonist SCH23390 (0.1 mg/kg) significantly disrupted the theta burst firing pattern. Administration of SCH23390 resulted in an overall significant variation in mean firing rate [F(3,13) = 5.31, P = 0.013] and the number of bursts/minute [F(3,13) = 3.96, P = 0.032]. There is a significant reduction of mean firing rate (P = 0.004) and number of bursts/minute (P = 0.016) at the 5–15 min postdrug injection epoch (Fig. 3D). Interestingly, in five of the eight cells tested for SCH23390, although their baseline responses showed regular theta frequency rhythmic bursts, they ceased to show rhythmic bursting after SCH23390.
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), and 5HT2C receptor activation also contributes to theta burst firing activity (Hajos et al. 2003). We thus tested the effects of a more selective D1/5 antagonist SCH39166 (McQuade et al. 1991). Intravenous SCH39166 (0.1 mg/kg) also abolished the baseline theta burst activity, but this antagonist induced a less marked reduction of the mean firing rate in MS/vDB neurons (n = 4, results not shown). It appears that a tonic D1/5 receptor mediated tone is regulating the theta burst frequency bursts in MS/vDB neurons. D1/5 Receptor activation mainly increases the mean firing rate, mean spike bursts, and mean spike per burst in Type 1, but not Type 2, MS/vDB neurons, and not MS/vDB-HIPP neurons
With the finding that D1/5 receptor blockade disrupts MS/vDB rhythmic theta burst firing patterns, we then attempted to determine whether D1/5 receptor activation could potentiate the firing rate and burst firing pattern of MS/vDB neurons. The baseline firing patterns of 39 MS/vDB neurons were monitored for 20–40 min, a time frame that appeared to be sufficient to establish a consistent pattern of overall firing characteristics. After an intravenous injection of the drug vehicle, firing activity of these MS/vDB neurons was recorded for another 20–40 min. This was followed by an intravenous injection of a D1 agonist dihydrexidine (DHX, 10 mg/kg) before an additional 20–40 min of postdrug response was recorded. Wherever cell recording quality could be maintained for an extended period of time, firing response to atropine (10 mg/kg iv) was also tested toward the end of the experiment to determine whether or not atropine disrupted theta burst firing and/or changes the firing rate (Stewart and Fox 1989c; Stewart et al. 1992). While it continues to be a subject of debate, atropine-sensitive MS/DB neurons have been proposed to be putative cholinergic neurons because simultaneous recordings of single MS/DB neurons and hippocampal EEG showed that atropine abolished theta burst firing along with the HIPP theta EEG (Brazhnik and Fox 1999; Stewart and Fox 1989c; Stewart et al. 1992).
In response to an intravenous injection of the D1 agonist DHX (10 mg/kg), the majority (n = 24; 60%) of MS/vDB neurons showed a slow sustained increase in mean firing rate, regardless whether they exhibit theta burst firing in the baseline or not (Fig. 4, A–C). In the theta bursting MS/vDB neurons, the increase in mean firing rate is also accompanied by an increase in the number of spike bursts as shown in the spike traces in Fig. 4A. Based on a DHX-induced ±20% change in mean firing rate compared with baseline mean firing rate, we categorized the D1/5 agonist-responsive MS/vDB neurons into two subtypes. In Type 1 D1-responsive MS/vDB neurons (n = 24), group data showed that there was a significant overall change in the mean firing rate [F(3,40) = 17.5; P = 0.0001; Fig. 4C]. Twenty-four Type 1 MS/vDB neurons (60%) showed a net (P < 0.05) significant increase in mean firing rate by 146 ± 35.5% compared with baseline. Fourteen of the Type 1 MS/vDB neurons were theta burst firing MS/vDB neurons, whereas the remaining 10 cells were non-theta MS-vDB cells.
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The second group of DHX-responsive cells are classified as Type 2 MS/vDB cells (n = 9, 23%), and their mean firing rate tends to be suppressed by intravenous DHX (-53 ± 11%, P > 0.05 compared with baseline) although there was no overall change in the group ANOVA analyses. Three of these nine Type 2 cells were antidromically activated putative MS/vDB-HIPP neurons, which showed a net reduction of –9.3 ± 9.3% (P > 0.05 compared with baseline). Like the Type-1 MS/vDB neurons, the mean firing rate of Type-2 neurons was unaffected by atropine administration. Seven MS/vDB neurons failed to respond to DHX (Fig. 4C).
We have also analyzed the change in the number of spike bursts as well as the mean number of spikes per burst before and after intravenous D1 agonist and atropine administration in both types of D1-responsive neurons (Fig. 4D). There was an overall significance [F(3,25) = 3.87, P = 0.021] in the group mean in the mean spike bursts for Type 1 MS/vDB neurons and a significant increase (P < 0.05) in the mean spike bursts over baseline mean in the MS/vDB neurons but not the antidromically activated MS/vDB-HIPP neurons. However, there was no overall change (P > 0.05) in the mean spike bursts in the Type 2 MS/vDB and MS/vDB-HIPP neurons (Fig. 4D).
We also analyzed mean spikes per burst in all MS/vDB neurons recorded. Group mean value show that there is a significant change in the treatments [F(3,39) = 11.77, P < 0.0001]. DHX significantly enhanced (P < 0.05) the mean number of spikes per burst in Type 1 MS/vDB neurons but not MS/vDB-HIPP neurons (Fig. 4E). For Type 2 MS/vDB or MS/vDB-HIPP neurons, there was no overall change (P > 0.05) in their mean spike bursts or mean spikes per burst. Thus it appears that the prominent significant change after D1/5 receptor stimulation is an increase in mean firing rate, number of bursts, and the number of spikes within each burst mainly in Type 1 MS/vDB neurons.
D1/5 receptor modulates the duration of time that MS/vDB neurons spent in a theta burst firing mode
We have also analyzed the percentage of time at which the MS/vDB neurons spent in theta burst firing mode—theta dwell time. This was accomplished by determining the total duration of occurrence of theta bursts by subjecting the spike data toautocorrelation analyses every 60 s for the entire period of continuous recording in each MS/vDB neuron before and after DHX, We called this the theta dwell time. Of the 40 cells analyzed, 19 (47.5%) showed a consistent basal rhythmic pattern of burst firing at theta frequencies (3–5 Hz, i.e., 3–5 spike density peaks in the autocorrelograms). ANOVA analysis of all (24) the Type 1 cell group showed that there was no overall change in the time that these cells spent in theta burst firing mode [F(3,23) = 1.34, P = 0.28] due to grouping together of both rhythmic theta bursting cells and nonrhythmic firing Type 1 MS/vDB neurons (Fig. 5E). It is notable that atropine treatment invariably abolished all theta burst activity in all individual Type 1 MS/vDB neurons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Our TH-immunocytochemical studies show under dark field microscopy that a sparse network of TH-stained axonal varicosities of different thickness is present in the MS/vDB. Previous double-labeling studies of anterograde tracer with TH immunoreactivity have shown that the MS/vDB does receive a sparse DA input from the VTA (Fallon and Moore 1978; Gaykema and Zaborszky 1996, 1997; Kalivas et al. 1985; Lindvall and Stenevi 1978; Moore 1978; Onteniente et al. 1984). At least some of the TH-immunoreactive innervations contact parvalbumin (putative GABAergic)- and ChAT (putative cholinergic)-immunoreactive neurons in MS/vDB (Gaspar et al. 1985; Gaykema and Zaborszky 1996, 1997; Kalivas et al. 1985). Some of these GABAergic or cholinergic neurons form the septo-hippocampal pathway that are the pacemakers for HIPP theta rhythm generation (Vertes et al. 2004).
Extracellular single-unit recordings from our present study were restricted to MS/vDB neurons only, with no neurons recorded in the horizontal limb of the DBB. Spontaneous theta burst firing activity in anesthetized animals are characteristically in the lower frequency range (3–5 Hz), whereas in unanesthetized animals, a broader theta frequency range (e.g., 5–12 Hz) is often observed (Apartis et al. 1998). In our urethan-anesthetized animals, the autocorrelogram spike density peaks show three to five peaks in theta burst firing cells, consistent with the anesthetic effects on theta burst frequency range (Brazhnik and Vinogradova 1986). MS/vDB neurons are also very sensitive to noxious tail-pinch stimuli as shown in earlier studies (Apartis et al. 1998; Dutar et al. 1985a). In addition, VTA train stimulation also induced transiently theta bursts in the otherwise random baseline bursting MS/vDB neurons. This suggests that DA (and possibly along with other transmitters) release from the VTA stimulation may modulate theta burst firing in some MS/vDB neurons. This is consistent with the finding of a putative DA projection from the VTA to the MS/vDB (Gaykema and Zaborszky 1996). Moreover, based on the sensitivity of these neurons to D1/5 antagonist SCH23390 and SCH39166, it appears that the spontaneous theta burst firing of these neurons is regulated tonically by endogenous DA acting on a D1/5 receptor in vivo. Hence, there is a sparse, but functionally relevant, dopaminergic innervation of the MS/vDB.
Were the systemically administered D1/5 agonist-induced electrophysiological changes of MS/vDB neurons mediated by D1/5 receptor on the MS/vDB neurons recorded? Previous autoradiographic studies using 3HSCH23390 binding failed to show much binding sites in this region of forebrain (Ariano et al. 1997; Dawson et al. 1986; Wamsley et al. 1989). However, the recent use of monoclonal antibodies for murine D1a and D1b receptors, as well as rat polyclonal antibodies, has enabled detection of a considerable presence of D1b (D5) receptor immunoreactivities in double-labeled cholinergic neurons from both the vertical and horizontal limbs of the DB in rats (Berlanga et al. 2005; Luedtke et al. 1999) in spite of the negative finding from an earlier study that used a different batch of polyclonal D1b antibody (Ariano et al. 1997). Thus the electrophysiological effects of the D1/5 agonist DHX shown in this study may be due to activation of D5 receptor in at least some of the MS/vDB neurons recorded.
The neurochemical identities of the recorded MS/vDB neurons were unknown. In theta bursting type 1 MS/vDB neurons (i.e., those neurons that show an increase in mean firing rate by DHX), atropine administration invariably disrupted any on-going theta burst firing activity with no further enhancement of post-DHX mean firing rates in these cells. Although atropine-insensitive theta burst neurons (with narrower spike width of 0.16–0.29 ms and putative GABAergic) are also recorded in the MS/vDB of urethan-anesthetized rats (Stewart and Fox 1989a), the longer spike width (0.3–0.5 ms) and atropine-sensitive MS/vDB neurons that we have recorded in our study are likely to be putative cholinergic neurons (Brazhnik and Fox 1999; Stewart and Fox 1989c). It should be noted that this interpretation has also been challenged after the discovery that MS/vDB-HIPP cholinergic collateral within the MS/vDB can also enhance the firing rate of MS/vDB-HIPP GABAergic neurons (Alreja et al. 2000). Thus atropine blockade of a tonic cholinergic regulation of theta bursting GABAergic MS/vDB-HIPP neurons can also disrupt the theta activity generated by the GABAergic, as well as cholinergic, MS/vDB neurons.
With regard to the spontaneous firing rates, D1/5 agonist treatment resulted in an increase in firing rate of both atropine-sensitive theta burst as well as non-theta MS/vDB Type 1 neurons. For Type 1 non-theta cells, despite their consistent elevation of mean firing rate by the D1/5 agonist, there was no change to the firing rate or pattern by atropine. These non-theta Type 1 neurons may represent noncholinergic (GABAergic/glutamatergic) MS/vDB neurons. Thus D1/5 receptor stimulation enhances the firing rate of both theta (putative cholinergic) and non-theta (putative GABAergic or glutamatergic) MS/vDB neurons.
Based on the autocorrelation analyses, the increase in mean firing rate was accompanied with more bursts and greater number of spikes per burst. However, the duration of time that theta bursts are persisting was highly variable. The majority of MS/vDB theta burst firing neurons show a reduction in the theta dwell time after D1/5 receptor stimulation despite an increase in spontaneous mean firing rate in these neurons. For those MS/vDB neurons that did not show a continuous theta burst activity in the baseline, they showed an increase in the theta dwell time after D1/5 receptor stimulation. The mechanism for D1/5 agonist modulation of the theta dwell time cannot be deduced from the finding of this in vivo study. Brain slice preparations of MS/vDB may help to enable one to determine the mechanism of D1 actions in theta modulation of specific MS/vDB neurons that can be identified immunohistochemically as cholinergic or GABAergic (Alreja et al. 2000; Borhegyi et al. 2004; Sotty et al. 2003).
Early biochemical studies showed that systemic injection of a mixed DA agonist apomorphine reduces, but intra-septal injection of a mixed DA antagonist haloperidol or 6OHDA DA depletion increases, ex vivo HIPP ACh turnover (Robinson et al. 1979; Yanai et al. 1993). These authors suggested that DA has an inhibitory action on septohippocampal cholinergic transmission. Using in vivo microdialysis techniques, several groups have shown that systemic injection of D1/5 agonists, or a prolonged (4 h) intra-dorsal HIPP, but not intra-lateral septal, infusion of a D1/5 agonist (SKF38393), led to an increase in HIPP ACh efflux. This increase was blocked by intra-dorsal hippocampal infusion of a D1/5 antagonist SCH23390 (Day and Fibiger 1994; Fitch et al. 2004; Hersi et al. 1995, 2000; Imperato et al. 1993; Laplante et al. 2004; Robinson et al. 1979). Recent data from the use of D5 receptor knockout mice suggest that it is the D5 receptor in dorsal HIPP that regulates the ACh release (Hersi et al. 2000).
In our present study, the systemic injection of the D1/5 agonist DHX induced an increase in mean firing rate in the majority of MS/vDB neurons, as well as increasing the theta burst firing in atropine-sensitive MS/vDB neurons. However, the firing rate of the antidromically activated MS/vDB-HIPP neurons failed to be increased by DHX, even though some of these neurons may be putative cholinergic MS/vDB-HIPP neurons (because their theta burst firing pattern were abolished by atropine). This suggests that D1/5 agonist stimulation did not strongly activate putative cholinergic MS/vDB-HIPP neurons. Thus it is difficult to establish a causal relationship between a D1/5 agonist-induced firing rate change of MS/vDB-HIPP neurons and increase in hipopocampal ACh efflux. On the other hand, MS/vDB neurons that were not activated antidromically but responded robustly (i.e., by increasing mean firing rate) to the D1/5 agonist stimulation could also be putative cholinergic MS/vDB-HIPP neurons that did not get proper antidromical activation because the stimulation electrode position in fimbria/fornix could not adequately activate all the topographical organized MS/vDB-HIPP axonal fibers (Nyakas et al. 1987).
The present data suggest that the action of D1/5 receptor activation may provide tonic firing to drive putative MS/vDB cholinergic and/or GABAergic to entrain HIPP theta rhythm to mediate multiple cognitive, attentional processing, and sensorimotor integrations (Bland and Oddie 2001; Bland et al. 1999; Smythe et al. 1992; Yoder and Pang 2005). A direct or indirect D1/5 receptor-mediated activation of cholinergic MS/vDB neurons could increase ACh release in the HIPP to provide a tonic depolarizing muscarinic drive to increase the neuronal excitability of HIPP pyramidal neurons and interneurons to modulate HIPP theta amplitude (Azouz et al. 1994; Bland and Oddie 2001; Chapman and Lacaille 1999; Figenschou et al. 1996). A concurrent D1/5 receptor-mediated activation of MS/vDB GABA output to HIPP may also disinhibit HIPP local interneuron circuits to modulate HIPP theta frequency (Chapman and Lacaille 1999; Lamsa et al. 2005; Toth et al. 1997; Yoder and Pang 2005). Thus for MS/vDB neurons that are in low theta burst firing state, an overall D1/5 receptor mediated facilitation of mean spontaneous firing and theta burst activities will contribute critically in HIPP theta generation (Miura et al. 1987), which is critical for theta-dependent synaptic plasticity in the HIPP (Huerta and Lisman 1995; Lisman and Otmakhova 2001) as well as attention and arousal that are required for learning and memory (Chudasama and Robbins 2004; Isaac and Berridge 2003). On the other hand, for MS/vDB neurons that are already firing in robust phasic theta bursts, activation of D1/5 receptor still enhances their overall spontaneous firing rate, but there is a reduction in the number of theta bursts, and this modulates the transition of HIPP theta activity to other frequencies or to a theta desynchronized state. Future concurrent HIPP EEG measurement should provide further insights. Although the detail synaptic network mechanisms of the preceding working model remained to be elucidated, the present data suggest that dopamine inputs to the MS/vDB neurons can provide a state-dependent bidirectional switch to regulate theta activity in the HIPP via D1/5 receptor activation (see Fig. 6).
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Address for reprint requests and other correspondence: C. R. Yang, Neuroscience Discovery, Eli Lilly & Co., Lilly Corporate Ctr., Indianapolis, IN 46285-0510 (E-mail: cyang{at}lilly.com)
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, 0.1 mg/kg). Note that there was a rapid reduction of the spopntanous firing rate over an extended period of time, and it slowly recovered back to baseline after 
25 min. B: autocorrelograms constructed from data taken at three 10-s time periods showing baseline strong theta burst firing of this MS/vDB neuron (1), peak suppressive response of the spontaneous activity and theta burst firing to D1 receptor blockade by SCH23390 (2), and recovery of the spontaneous firing and theta burst activity (3). C: corresponding voltage traces of the spontaneous spike firing showing the theta burst pattern of firing taken in periods 1–3 when the autocorrelograms were constructed in B). D: group data summary in histograms showing a significant reduction (mainly in the 1st 5–10 min after SHC23390 adminstration) of both the mean firing rate and mean number of bursts per min. in the MS/vDB neurons analyzed.
20% after the D1 agonist DHX treatment, and they represent the majority of the neurons studied. DHX-insensitive cells are excluded in this analysis. Nonantidromic MS/vDB neurons are shown in open bars and antidromic MS/vDB-HIPP neurons are in gray bars. C–E, left: summarized the spike analyses group data, which show that the D1 agonist DHX increased the mean firing rate and the mean spike per burst in the MS/vDB neurons. Because of the high variability, DHX did not induce a significant change in the mean number of spike bursts. Note also that although atropine also shows a parallel increase in the firing rate and bursts when compared with the baseline, these changes were not different from the DHX responses. Right: Type 2 neurons are neurons whose mean firing rates were reduced by 
